Tinghu Zhang

All Publications

• Discovery of bivalent small molecule degraders of cyclin-dependent kinase 7 (CDK7). European journal of medicinal chemistry Ji, W., Du, G., Jiang, J., Lu, W., Mills, C. E., Yuan, L., Jiang, F., He, Z., Bradshaw, G. A., Chung, M., Jiang, Z., Byun, W. S., Hinshaw, S. M., Zhang, T., Gray, N. S. 2024; 276: 116613

  Abstract

  Cyclin-dependent kinase 7, along with cyclin H and MAT1, forms the CDK-activating complex (CAK), which directs cell cycle progression via T-loop phosphorylation of cell cycle CDKs. Pharmacological inhibition of CDK7 leads to selective anti-cancer effects in cellular and in vivo models, motivating several ongoing clinical investigations of this target. Current CDK7 inhibitors are either reversible or covalent inhibitors of its catalytic activity. We hypothesized that small molecule targeted protein degradation (TPD) might result in differentiated pharmacology due to the loss of scaffolding functions. Here, we report the design and characterization of a potent CDK7 degrader that is comprised of an ATP-competitive CDK7 binder linked to a CRL2VHL recruiter. JWZ-5-13 effectively degrades CDK7 in multiple cancer cells and leads to a potent inhibition of cell proliferation. Additionally, compound JWZ-5-13 displayed bioavailability in a pharmacokinetic study conducted in mice. Therefore, JWZ-5-13 is a useful chemical probe to investigate the pharmacological consequences of CDK7 degradation.

  View details for DOI 10.1016/j.ejmech.2024.116613

  View details for PubMedID 39004018

• Molecular Bidents with Two Electrophilic Warheads as a New Pharmacological Modality. ACS central science Li, Z., Jiang, J., Ficarro, S. B., Beyett, T. S., To, C., Tavares, I., Zhu, Y., Li, J., Eck, M. J., Jänne, P. A., Marto, J. A., Zhang, T., Che, J., Gray, N. S. 2024; 10 (6): 1156-1166

  Abstract

  A systematic strategy to develop dual-warhead inhibitors is introduced to circumvent the limitations of conventional covalent inhibitors such as vulnerability to mutations of the corresponding nucleophilic residue. Currently, all FDA-approved covalent small molecules feature one electrophile, leaving open a facile route to acquired resistance. We conducted a systematic analysis of human proteins in the protein data bank to reveal ∼400 unique targets amendable to dual covalent inhibitors, which we term "molecular bidents". We demonstrated this strategy by targeting two kinases: MKK7 and EGFR. The designed compounds, ZNL-8162 and ZNL-0056, are ATP-competitive inhibitors that form two covalent bonds with cysteines and retain potency against single cysteine mutants. Therefore, molecular bidents represent a new pharmacological modality with the potential for improved selectivity, potency, and drug resistance profile.

  View details for DOI 10.1021/acscentsci.3c01245

  View details for PubMedID 38947214

  View details for PubMedCentralID PMC11212140

• Broad-spectrum activity against mosquito-borne flaviviruses achieved by a targeted protein degradation mechanism. Nature communications Liu, H. Y., Li, Z., Reindl, T., He, Z., Qiu, X., Golden, R. P., Donovan, K. A., Bailey, A., Fischer, E. S., Zhang, T., Gray, N. S., Yang, P. L. 2024; 15 (1): 5179

  Abstract

  Viral genetic diversity presents significant challenges in developing antivirals with broad-spectrum activity and high barriers to resistance. Here we report development of proteolysis targeting chimeras (PROTACs) targeting the dengue virus envelope (E) protein through coupling of known E fusion inhibitors to ligands of the CRL4CRBN E3 ubiquitin ligase. The resulting small molecules block viral entry through inhibition of E-mediated membrane fusion and interfere with viral particle production by depleting intracellular E in infected Huh 7.5 cells. This activity is retained in the presence of point mutations previously shown to confer partial resistance to the parental inhibitors due to decreased inhibitor-binding. The E PROTACs also exhibit broadened spectrum of activity compared to the parental E inhibitors against a panel of mosquito-borne flaviviruses. These findings encourage further exploration of targeted protein degradation as a differentiated and potentially advantageous modality for development of broad-spectrum direct-acting antivirals.

  View details for DOI 10.1038/s41467-024-49161-9

  View details for PubMedID 38898037

  View details for PubMedCentralID PMC11187112

• ZNL0325, a Pyrazolopyrimidine-Based Covalent Probe, Demonstrates an Alternative Binding Mode for Kinases. Journal of medicinal chemistry Li, Z., Lu, W., Beyett, T. S., Ficarro, S. B., Jiang, J., Tse, J., Kim, A. Y., Marto, J. A., Che, J., Jänne, P. A., Eck, M. J., Zhang, T., Gray, N. S. 2024

  Abstract

  The pyrazolopyrimidine (PP) heterocycle is a versatile and widely deployed core scaffold for the development of kinase inhibitors. Typically, a 4-amino-substituted pyrazolopyrimidine binds in the ATP-binding pocket in a conformation analogous to the 6-aminopurine of ATP. Here, we report the discovery of ZNL0325 which exhibits a flipped binding mode where the C3 position is oriented toward the ribose binding pocket. ZNL0325 and its analogues feature an acrylamide side chain at the C3 position which is capable of forming a covalent bond with multiple kinases that possess a cysteine at the αD-1 position including BTK, EGFR, BLK, and JAK3. These findings suggest that the ability to form a covalent bond can override the preferred noncovalent binding conformation of the heterocyclic core and provides an opportunity to create structurally distinct covalent kinase inhibitors.

  View details for DOI 10.1021/acs.jmedchem.3c01891

  View details for PubMedID 38300264

• Genome-Wide CRISPR Screens Identify Multiple Synthetic Lethal Targets That Enhance KRASG12C Inhibitor Efficacy. Cancer research Mukhopadhyay, S., Huang, H. Y., Lin, Z., Ranieri, M., Li, S., Sahu, S., Liu, Y., Ban, Y., Guidry, K., Hu, H., Lopez, A., Sherman, F., Tan, Y. J., Lee, Y. T., Armstrong, A. P., Dolgalev, I., Sahu, P., Zhang, T., Lu, W., Gray, N. S., Christensen, J. G., Tang, T. T., Velcheti, V., Khodadadi-Jamayran, A., Wong, K. K., Neel, B. G. 2023

  Abstract

  Non-small lung cancers (NSCLCs) frequently (~30%) harbor KRAS driver mutations, half of which are KRASG12C. KRAS-mutant NSCLC with co-mutated STK11 and/or KEAP1 is particularly refractory to conventional, targeted, and immune therapy. Development of KRASG12C inhibitors (G12Cis) provided a major therapeutic advance, but resistance still limits their efficacy. To identify genes whose deletion augments efficacy of the G12Cis adagrasib (MRTX-849) or adagrasib plus TNO155 (SHP2i), we performed genome-wide CRISPR/Cas9 screens on KRAS/STK11-mutant NSCLC lines. Recurrent, potentially targetable, synthetic lethal (SL) genes were identified, including serine-threonine kinases, tRNA-modifying and proteoglycan synthesis enzymes, and YAP/TAZ/TEAD pathway components. Several SL genes were confirmed by siRNA/shRNA experiments, and the YAP/TAZ/TEAD pathway was extensively validated in vitro and in mice. Mechanistic studies showed that G12Ci treatment induced gene expression of RHO paralogs and activators, increased RHOA activation, and evoked ROCK-dependent nuclear translocation of YAP. Mice and patients with acquired G12Ci- or G12Ci/SHP2i-resistant tumors showed strong overlap with SL pathways, arguing for the relevance of the screen results. These findings provide a landscape of potential targets for future combination strategies, some of which can be tested rapidly in the clinic.

  View details for DOI 10.1158/0008-5472.CAN-23-2729

  View details for PubMedID 37729426

• Proteomics-Based Discovery of First-in-Class Chemical Probes for Programmed Cell Death Protein 2 (PDCD2). Angewandte Chemie (International ed. in English) Ji, W., Byun, W. S., Lu, W., Zhu, X., Donovan, K. A., Dwyer, B., Che, J., Yuan, L., Abulaiti, X., Corsello, S. M., Fischer, E. S., Zhang, T., Gray, N. S. 2023: e202308292

  Abstract

  Chemical probes are essential tools for understanding biological systems and for credentialing potential biomedical targets. Programmed cell death 2 (PDCD2) is a member of the B-cell lymphoma 2 (Bcl-2) family of proteins, which are critical regulators of apoptosis. Here we report the discovery and characterization of 10e, a first-in-class small molecule degrader of PDCD2. We discovered PDCD2 degrader by serendipity using a chemical proteomics approach in contrast to the conventional approach for making bivalent degraders starting from a known binding ligand targeting the protein of interest. Using 10e as a pharmacological probe, we demonstrate that PDCD2 functions as a critical regulator of cell growth by modulating the progression of the cell cycle in T lymphoblasts. Our work provides a useful pharmacological probe for investigating PDCD2 function and highlights using chemical proteomics to discover selective small molecule degraders of unanticipated targets.

  View details for DOI 10.1002/anie.202308292

  View details for PubMedID 37658265

• Rewiring cancer drivers to activate apoptosis. Nature Gourisankar, S., Krokhotin, A., Ji, W., Liu, X., Chang, C., Kim, S. H., Li, Z., Wenderski, W., Simanauskaite, J. M., Yang, H., Vogel, H., Zhang, T., Green, M. R., Gray, N. S., Crabtree, G. R. 2023

  Abstract

  Genes that drive the proliferation, survival, invasion and metastasis of malignant cells have been identified for many human cancers1-4. Independent studies have identified cell death pathways that eliminate cells for the good of the organism5,6. The coexistence of cell death pathways with driver mutations suggests that the cancer driver could be rewired to activate cell death using chemical inducers of proximity (CIPs). Here we describe a new class of molecules called transcriptional/epigenetic CIPs (TCIPs) that recruit the endogenous cancer driver, or a downstream transcription factor, to the promoters of cell death genes, thereby activating their expression. We focused on diffuse large B cell lymphoma, in which the transcription factor B cell lymphoma 6 (BCL6) is deregulated7. BCL6 binds to the promoters of cell death genes and epigenetically suppresses their expression8. We produced TCIPs by covalently linking small molecules that bind BCL6 to those that bind to transcriptional activators that contribute to the oncogenic program, such as BRD4. The most potent molecule, TCIP1, increases binding of BRD4 by 50% over genomic BCL6-binding sites to produce transcriptional elongation at pro-apoptotic target genes within 15min, while reducing binding of BRD4 over enhancers by only 10%, reflecting a gain-of-function mechanism. TCIP1 kills diffuse large B cell lymphoma cell lines, including chemotherapy-resistant, TP53-mutant lines, at EC50 of 1-10nM in 72h and exhibits cell-specific and tissue-specific effects, capturing the combinatorial specificity inherent to transcription. The TCIP concept also has therapeutic applications in regulating the expression of genes for regenerative medicine and developmental disorders.

  View details for DOI 10.1038/s41586-023-06348-2

  View details for PubMedID 37495688

• New scaffolds for type II JAK2 inhibitors overcome the acquired G993A resistance mutation. Cell chemical biology Arwood, M. L., Liu, Y., Harkins, S. K., Weinstock, D. M., Yang, L., Stevenson, K. E., Plana, O. D., Dong, J., Cirka, H., Jones, K. L., Virtanen, A. T., Gupta, D. G., Ceas, A., Lawney, B., Yoda, A., Leahy, C., Hao, M., He, Z., Choi, H. G., Wang, Y., Silvennoinen, O., Hubbard, S. R., Zhang, T., Gray, N. S., Li, L. S. 2023

  Abstract

  Recurrent JAK2 alterations are observed in myeloproliferative neoplasms, B-cell acute lymphoblastic leukemia, and other hematologic malignancies. Currently available type I JAK2 inhibitors have limited activity in these diseases. Preclinical data support the improved efficacy of type II JAK2 inhibitors, which lock the kinase in the inactive conformation. By screening small molecule libraries, we identified a lead compound with JAK2 selectivity. We highlight analogs with on-target biochemical and cellular activity and demonstrate in vivo activity using a mouse model of polycythemia vera. We present a co-crystal structure that confirms the type II binding mode of our compounds with the "DFG-out" conformation of the JAK2 activation loop. Finally, we identify a JAK2 G993A mutation that confers resistance to the type II JAK2 inhibitor CHZ868 but not to our analogs. These data provide a template for identifying novel type II kinase inhibitors and inform further development of agents targeting JAK2 that overcome resistance.

  View details for DOI 10.1016/j.chembiol.2023.05.007

  View details for PubMedID 37290440

• Development and characterization of selective FAK inhibitors and PROTACs with in vivo activity. Chembiochem : a European journal of chemical biology Koide, E., Mohardt, M. L., Doctor, Z. M., Yang, A., Hao, M., Donovan, K. A., Kuismi, C. C., Nelson, A. J., Abell, K., Aguiar, M., Che, J., Stokes, M. P., Zhang, T., Aguirre, A. J., Fischer, E. S., Gray, N. S., Jiang, B., Nabet, B. 2023: e202300141

  Abstract

  Focal adhesion kinase (FAK) is an attractive drug target due to its overexpression in cancer. FAK functions as a non-receptor tyrosine kinase and scaffolding protein, coordinating several downstream signaling effectors and cellular processes. While drug discovery efforts have largely focused on targeting FAK kinase activity, FAK inhibitors have failed to show efficacy as single agents in clinical trials. Here, using structure-guided design, we report the development of a selective FAK inhibitor (BSJ-04-175) and degrader (BSJ-04-146) to evaluate the consequences and advantages of abolishing all FAK activity in cancer models. BSJ-04-146 achieves rapid and potent FAK degradation with high proteome-wide specificity in cancer cells and induces durable degradation in mice. Compared to kinase inhibition, targeted degradation of FAK exhibits pronounced improved activity on downstream signaling and cancer cell viability and migration. Together, BSJ-04-175 and BSJ-04-146 are valuable chemical tools to dissect the specific consequences of targeting FAK through small molecule inhibition or degradation.

  View details for DOI 10.1002/cbic.202300141

  View details for PubMedID 37088717

• Catalytic Degraders Effectively Address Kinase Site Mutations in EML4-ALK Oncogenic Fusions. Journal of medicinal chemistry Gao, Y., Jiang, B., Kim, H., Berberich, M. J., Che, J., Donovan, K. A., Hatcher, J. M., Huerta, F., Kwiatkowski, N. P., Liu, Y., Liuni, P. P., Metivier, R. J., Murali, V. K., Nowak, R. P., Zhang, T., Fischer, E. S., Gray, N. S., Jones, L. H. 2023

  Abstract

  Heterobifunctional degraders, known as proteolysis targeting chimeras (PROTACs), theoretically possess a catalytic mode-of-action, yet few studies have either confirmed or exploited this potential advantage of event-driven pharmacology. Degraders of oncogenic EML4-ALK fusions were developed by conjugating ALK inhibitors to cereblon ligands. Simultaneous optimization of pharmacology and compound properties using ternary complex modeling and physicochemical considerations yielded multiple catalytic degraders that were more resilient to clinically relevant ATP-binding site mutations than kinase inhibitor drugs. Our strategy culminated in the design of the orally bioavailable derivative CPD-1224 that avoided hemolysis (a feature of detergent-like PROTACs), degraded the otherwise recalcitrant mutant L1196M/G1202R in vivo, and commensurately slowed tumor growth, while the third generation ALK inhibitor drug lorlatinib had no effect. These results validate our original therapeutic hypothesis by exemplifying opportunities for catalytic degraders to proactively address binding site resistant mutations in cancer.

  View details for DOI 10.1021/acs.jmedchem.2c01864

  View details for PubMedID 37036171

• Structure-Based Design of Y-Shaped Covalent TEAD Inhibitors. Journal of medicinal chemistry Lu, W., Fan, M., Ji, W., Tse, J., You, I., Ficarro, S. B., Tavares, I., Che, J., Kim, A. Y., Zhu, X., Boghossian, A., Rees, M. G., Ronan, M. M., Roth, J. A., Hinshaw, S. M., Nabet, B., Corsello, S. M., Kwiatkowski, N., Marto, J. A., Zhang, T., Gray, N. S. 2023

  Abstract

  Transcriptional enhanced associate domain (TEAD) proteins together with their transcriptional coactivator yes-associated protein (YAP) and transcriptional coactivator with the PDZ-binding motif (TAZ) are important transcription factors and cofactors that regulate gene expression in the Hippo pathway. In mammals, the TEAD families have four homologues: TEAD1 (TEF-1), TEAD2 (TEF-4), TEAD3 (TEF-5), and TEAD4 (TEF-3). Aberrant expression and hyperactivation of TEAD/YAP signaling have been implicated in a variety of malignancies. Recently, TEADs were recognized as being palmitoylated in cells, and the lipophilic palmitate pocket has been successfully targeted by both covalent and noncovalent ligands. In this report, we present the medicinal chemistry effort to develop MYF-03-176 (compound 22) as a selective, cysteine-covalent TEAD inhibitor. MYF-03-176 (compound 22) significantly inhibits TEAD-regulated gene expression and proliferation of the cell lines with TEAD dependence including those derived from mesothelioma and liposarcoma.

  View details for DOI 10.1021/acs.jmedchem.2c01548

  View details for PubMedID 36946421

• Development of a Covalent Inhibitor of c-Jun N-Terminal Protein Kinase (JNK) 2/3 with Selectivity over JNK1. Journal of medicinal chemistry Lu, W., Liu, Y., Gao, Y., Geng, Q., Gurbani, D., Li, L., Ficarro, S. B., Meyer, C. J., Sinha, D., You, I., Tse, J., He, Z., Ji, W., Che, J., Kim, A. Y., Yu, T., Wen, K., Anderson, K. C., Marto, J. A., Westover, K. D., Zhang, T., Gray, N. S. 2023

  Abstract

  The c-Jun N-terminal kinases (JNKs) are members of the mitogen-activated protein kinase (MAPK) family, which includes JNK1-JNK3. Interestingly, JNK1 and JNK2 show opposing functions, with JNK2 activity favoring cell survival and JNK1 stimulating apoptosis. Isoform-selective small molecule inhibitors of JNK1 or JNK2 would be useful as pharmacological probes but have been difficult to develop due to the similarity of their ATP binding pockets. Here, we describe the discovery of a covalent inhibitor YL5084, the first such inhibitor that displays selectivity for JNK2 over JNK1. We demonstrated that YL5084 forms a covalent bond with Cys116 of JNK2, exhibits a 20-fold higher Kinact/KI compared to that of JNK1, and engages JNK2 in cells. However, YL5084 exhibited JNK2-independent antiproliferative effects in multiple myeloma cells, suggesting the existence of additional targets relevant in this context. Thus, although not fully optimized, YL5084 represents a useful chemical starting point for the future development of JNK2-selective chemical probes.

  View details for DOI 10.1021/acs.jmedchem.2c01834

  View details for PubMedID 36826833

• Depletion of creatine phosphagen energetics with a covalent creatine kinase inhibitor. Nature chemical biology Darabedian, N., Ji, W., Fan, M., Lin, S., Seo, H. S., Vinogradova, E. V., Yaron, T. M., Mills, E. L., Xiao, H., Senkane, K., Huntsman, E. M., Johnson, J. L., Che, J., Cantley, L. C., Cravatt, B. F., Dhe-Paganon, S., Stegmaier, K., Zhang, T., Gray, N. S., Chouchani, E. T. 2023

  Abstract

  Creatine kinases (CKs) provide local ATP production in periods of elevated energetic demand, such as during rapid anabolism and growth. Thus, creatine energetics has emerged as a major metabolic liability in many rapidly proliferating cancers. Whether CKs can be targeted therapeutically is unknown because no potent or selective CK inhibitors have been developed. Here we leverage an active site cysteine present in all CK isoforms to develop a selective covalent inhibitor of creatine phosphagen energetics, CKi. Using deep chemoproteomics, we discover that CKi selectively engages the active site cysteine of CKs in cells. A co-crystal structure of CKi with creatine kinase B indicates active site inhibition that prevents bidirectional phosphotransfer. In cells, CKi and its analogs rapidly and selectively deplete creatine phosphate, and drive toxicity selectively in CK-dependent acute myeloid leukemia. Finally, we use CKi to uncover an essential role for CKs in the regulation of proinflammatory cytokine production in macrophages.

  View details for DOI 10.1038/s41589-023-01273-x

  View details for PubMedID 36823351

  View details for PubMedCentralID 5540325

• Development of potent and selective degraders of PI5P4Kgamma. European journal of medicinal chemistry Ji, W., Wang, E. S., Manz, T. D., Jiang, J., Donovan, K. A., Abulaiti, X., Fischer, E. S., Cantley, L. C., Zhang, T., Gray, N. S. 2022; 247: 115027

  Abstract

  Phosphatidylinositol 5-phosphate 4-kinases (PI5P4Ks), a family of three members in mammals (alpha, beta and gamma), have emerged as potential therapeutic targets due to their role in regulating many important cellular signaling pathways. In comparison to the PI5P4Kalpha and PI5P4Kbeta, which usually have similar expression profiles across cancer cells, PI5P4Kgamma exhibits distinct expression patterns, and pathological functions for PI5P4Kgamma have been proposed in the context of cancer and neurodegenerative diseases. PI5P4Kgamma has very low kinase activity and has been proposed to inhibit the PI4P5Ks through scaffolding function, providing a rationale for developing a selective PI5P4Kgamma degrader. Here, we report the development and characterization of JWZ-1-80, a first-in-class PI5P4Kgamma degrader. JWZ-1-80 potently degrades PI5P4Kgamma via the ubiquitin-proteasome system and exhibits proteome-wide selectivity and is therefore a useful tool compound for further dissecting the biological functions of PI5P4Kgamma.

  View details for DOI 10.1016/j.ejmech.2022.115027

  View details for PubMedID 36584631

• Covalent disruptor of YAP-TEAD association suppresses defective hippo signaling. eLife Fan, M., Lu, W., Che, J., Kwiatkowski, N. P., Gao, Y., Seo, H., Ficarro, S. B., Gokhale, P. C., Liu, Y., Geffken, E. A., Lakhani, J., Song, K., Kuljanin, M., Ji, W., Jiang, J., He, Z., Tse, J., Boghossian, A. S., Rees, M. G., Ronan, M. M., Roth, J. A., Mancias, J. D., Marto, J. A., Dhe-Paganon, S., Zhang, T., Gray, N. S. 2022; 11

  Abstract

  The transcription factor TEAD, together with its coactivator YAP/TAZ, is a key transcriptional modulator of the Hippo pathway. Activation of TEAD transcription by YAP has been implicated in a number of malignancies, and this complex represents a promising target for drug discovery. However, both YAP and its extensive binding interfaces to TEAD have been difficult to address using small molecules, mainly due to a lack of druggable pockets. TEAD is post-translationally modified by palmitoylation that targets a conserved cysteine at a central pocket, which provides an opportunity to develop cysteine-directed covalent small molecules for TEAD inhibition. Here, we employed covalent fragment screening approach followed by structure-based design to develop an irreversible TEAD inhibitor MYF-03-69. Using a range of in vitro and cell-based assays we demonstrated that through a covalent binding with TEAD palmitate pocket, MYF-03-69 disrupts YAP-TEAD association, suppresses TEAD transcriptional activity and inhibits cell growth of Hippo signaling defective malignant pleural mesothelioma (MPM). Further, a cell viability screening with a panel of 903 cancer cell lines indicated a high correlation between TEAD-YAP dependency and the sensitivity to MYF-03-69. Transcription profiling identified the upregulation of proapoptotic BMF gene in cancer cells that are sensitive to TEAD inhibition. Further optimization of MYF-03-69 led to an in vivo compatible compound MYF-03-176, which shows strong antitumor efficacy in MPM mouse xenograft model via oral administration. Taken together, we disclosed a story of the development of covalent TEAD inhibitors and its high therapeutic potential for clinic treatment for the cancers that are driven by TEAD-YAP alteration.

  View details for DOI 10.7554/eLife.78810

  View details for PubMedID 36300789

• Exploring the target scope of KEAP1 E3 ligase-based PROTACs. Cell chemical biology Du, G., Jiang, J., Henning, N. J., Safaee, N., Koide, E., Nowak, R. P., Donovan, K. A., Yoon, H., You, I., Yue, H., Eleuteri, N. A., He, Z., Li, Z., Huang, H. T., Che, J., Nabet, B., Zhang, T., Fischer, E. S., Gray, N. S. 2022

  Abstract

  Targeted protein degradation (TPD) uses small molecules to recruit E3 ubiquitin ligases into the proximity of proteins of interest, inducing ubiquitination-dependent degradation. A major bottleneck in the TPD field is the lack of accessible E3 ligase ligands for developing degraders. To expand the E3 ligase toolbox, we sought to convert the Kelch-like ECH-associated protein 1 (KEAP1) inhibitor KI696 into a recruitment handle for several targets. While we were able to generate KEAP1-recruiting degraders of BET family and murine focal adhesion kinase (FAK), we discovered that the target scope of KEAP1 was narrow, as targets easily degraded using a cereblon (CRBN)-recruiting degrader were refractory to KEAP1-mediated degradation. Linking the KEAP1-binding ligand to a CRBN-binding ligand resulted in a molecule that induced degradation of KEAP1 but not CRBN. In sum, we characterize tool compounds to explore KEAP1-mediated ubiquitination and delineate the challenges of exploiting new E3 ligases for generating bivalent degraders.

  View details for DOI 10.1016/j.chembiol.2022.08.003

  View details for PubMedID 36070758

• Targeting transcription in heart failure via CDK7/12/13 inhibition. Nature communications Hsu, A., Duan, Q., Day, D. S., Luo, X., McMahon, S., Huang, Y., Feldman, Z. B., Jiang, Z., Zhang, T., Liang, Y., Alexanian, M., Padmanabhan, A., Brown, J. D., Lin, C. Y., Gray, N. S., Young, R. A., Bruneau, B. G., Haldar, S. M. 2022; 13 (1): 4345

  Abstract

  Heart failure with reduced ejection fraction (HFrEF) is associated with high mortality, highlighting an urgent need for new therapeutic strategies. As stress-activated cardiac signaling cascades converge on the nucleus to drive maladaptive gene programs, interdicting pathological transcription is a conceptually attractive approach for HFrEF therapy. Here, we demonstrate that CDK7/12/13 are critical regulators of transcription activation in the heart that can be pharmacologically inhibited to improve HFrEF. CDK7/12/13 inhibition using the first-in-class inhibitor THZ1 or RNAi blocks stress-induced transcription and pathologic hypertrophy in cultured rodent cardiomyocytes. THZ1 potently attenuates adverse cardiac remodeling and HFrEF pathogenesis in mice and blocks cardinal features of disease in human iPSC-derived cardiomyocytes. THZ1 suppresses Pol II enrichment at stress-transactivated cardiac genes and inhibits a specific pathologic gene program in the failing mouse heart. These data identify CDK7/12/13 as druggable regulators of cardiac gene transactivation during disease-related stress, suggesting that HFrEF features a critical dependency on transcription that can be therapeutically exploited.

  View details for DOI 10.1038/s41467-022-31541-8

  View details for PubMedID 35896549

• Synthesis and Structure-Activity relationships of cyclin-dependent kinase 11 inhibitors based on a diaminothiazole scaffold. European journal of medicinal chemistry Li, Z., Ishida, R., Liu, Y., Wang, J., Li, Y., Gao, Y., Jiang, J., Che, J., Sheltzer, J. M., Robers, M. B., Zhang, T., Westover, K. D., Nabet, B., Gray, N. S. 2022; 238: 114433

  Abstract

  Cyclin-dependent kinases (CDK) are attractive targets for drug discovery due to their wide range of cellular functions. CDK11 is an understudied CDK with roles in transcription and splicing, cell cycle regulation, neuronal function, and apoptosis. In this study, we describe a medicinal chemistry campaign to identify a CDK11 inhibitor. Employing a promising but nonselective CDK11-targeting scaffold (JWD-047), extensive structure-guided medicinal chemistry modifications led to the identification of ZNL-05-044. A combination of biochemical evaluations and NanoBRET cellular assays for target engagement guided the SAR towards a 2,4-diaminothiazoles CDK11 probe with significantly improved kinome-wide selectivity over JWD-047. CDK11 inhibition with ZNL-05-044 leads to G2/M cell cycle arrest, consistent with prior work evaluating OTS964, and impacts CDK11-dependent mRNA splicing in cells. Together, ZNL-05-044 serves as a tool compound for further optimization and interrogation of the consequences of CDK11 inhibition.

  View details for DOI 10.1016/j.ejmech.2022.114433

  View details for PubMedID 35597007

• A preclinical platform for assessing antitumor effects and systemic toxicities of cancer drug targets. Proceedings of the National Academy of Sciences of the United States of America Li, X., Huang, C. H., Sánchez-Rivera, F. J., Kennedy, M. C., Tschaharganeh, D. F., Morris, J. P., Montinaro, A., O'Rourke, K. P., Banito, A., Wilkinson, J. E., Chen, C. C., Ho, Y. J., Dow, L. E., Tian, S., Luan, W., de Stanchina, E., Zhang, T., Gray, N. S., Walczak, H., Lowe, S. W. 2022; 119 (17): e2110557119

  Abstract

  SignificanceMany new cancer drugs fail at the clinical stage owing to poor efficacy and/or excessive toxicity, though whether this reflects shortcomings of the target or the drug is often unclear. To gain earlier insights into factors that can influence the therapeutic index of target inhibition in vivo, we combine inducible RNA interference and somatic engineering technologies to produce a cost-effective platform that enables systemic and inducible suppression of candidate target in normal tissues and tumor cells in the same mouse. By comparing the consequences of genetic and pharmacological CDK9 inhibition, we establish the utility of this platform to predict factors influencing the therapeutic index. Additionally, our studies provide support, and some cautionary notes, for the clinical development of CDK9 inhibitors.

  View details for DOI 10.1073/pnas.2110557119

  View details for PubMedID 35442775

• Development of PDE6D and CK1alpha Degraders through Chemical Derivatization of FPFT-2216. Journal of medicinal chemistry Teng, M., Lu, W., Donovan, K. A., Sun, J., Krupnick, N. M., Nowak, R. P., Li, Y., Sperling, A. S., Zhang, T., Ebert, B. L., Fischer, E. S., Gray, N. S. 1800

  Abstract

  Immunomodulatory drugs are a class of drugs approved for the treatment of multiple myeloma. These compounds exert their clinical effects by inducing interactions between the CRL4CRBN E3 ubiquitin ligase and a C2H2 zinc finger degron motif, resulting in degradation of degron-containing targets. However, although many cellular proteins feature the degron motif, only a subset of those are degradable via this strategy. Here, we demonstrated that FPFT-2216, a previously reported "molecular glue" compound, degrades PDE6D, in addition to IKZF1, IKZF3, and CK1alpha. We used FPFT-2216 as a starting point for a focused medicinal chemistry campaign and developed TMX-4100 and TMX-4116, which exhibit greater selectivity for degrading PDE6D and CK1alpha, respectively. We also showed that the region in PDE6D that interacts with the FPFT-2216 derivatives is not the previously pursued prenyl-binding pocket. Moreover, we found that PDE6D depletion by FPFT-2216 does not impede the growth of KRASG12C-dependent MIA PaCa-2 cells, highlighting the challenges of drugging PDE6D-KRAS. Taken together, the approach we described here represents a general scheme to rapidly develop selective degraders by reprogramming E3 ubiquitin ligase substrate specificity.

  View details for DOI 10.1021/acs.jmedchem.1c01832

  View details for PubMedID 34965125

• Structure-activity relationship study of THZ531 derivatives enables the discovery of BSJ-01-175 as a dual CDK12/13 covalent inhibitor with efficacy in Ewing sarcoma. European journal of medicinal chemistry Jiang, B., Jiang, J., Kaltheuner, I. H., Iniguez, A. B., Anand, K., Ferguson, F. M., Ficarro, S. B., Seong, B. K., Greifenberg, A. K., Dust, S., Kwiatkowski, N. P., Marto, J. A., Stegmaier, K., Zhang, T., Geyer, M., Gray, N. S. 2021; 221: 113481

  Abstract

  Development of inhibitors targeting CDK12/13 is of increasing interest as a potential therapy for cancers as these compounds inhibit transcription of DNA damage response (DDR) genes. We previously described THZ531, a covalent inhibitor with selectivity for CDK12/13. In order to elucidate structure-activity relationship (SAR), we have undertaken a medicinal chemistry campaign and established a focused library of THZ531 analogs. Among these analogs, BSJ-01-175 demonstrates exquisite selectivity, potent inhibition of RNA polymerase II phosphorylation, and downregulation of CDK12-targeted genes in cancer cells. A 3.0 Å co-crystal structure with CDK12/CycK provides a structural rational for selective targeting of Cys1039 located in a C-terminal extension from the kinase domain. With moderate pharmacokinetic properties, BSJ-01-175 exhibits efficacy against an Ewing sarcoma tumor growth in a patient-derived xenograft (PDX) mouse model following 10 mg/kg once a day, intraperitoneal administration. Taken together, BSJ-01-175 represents the first selective CDK12/13 covalent inhibitor with in vivo efficacy reported to date.

  View details for DOI 10.1016/j.ejmech.2021.113481

  View details for PubMedID 33945934

• Selective degradation-inducing probes for studying cereblon (CRBN) biology. RSC medicinal chemistry Powell, C. E., Du, G., Bushman, J. W., He, Z., Zhang, T., Fischer, E. S., Gray, N. S. 2021; 12 (8): 1381-1390

  Abstract

  Targeted protein degradation represents a rapidly growing area in drug discovery and development. Moreover, small molecules that induce the targeted degradation of a given protein also represent an important addition to the chemical probes toolbox as these compounds can achieve selective protein knockdown, thus providing an approach that is orthogonal to genetic knockdowns. In order to develop degradation-inducing chemical probes for studying cereblon (CRBN) biology, we generated six CRBN-CRBN (homo-PROTAC) degraders and six CRBN-VHL (hetero-PROTAC) degraders. From these compounds we identified two potent and selective CRBN degraders (ZXH-4-130 and ZXH-4-137), both of which are CRBN-VHL compounds. We characterized these lead degraders by quantitative proteomics in five cell lines (MM1.S, Kelly, SK-N-DZ, HEK293T, and MOLT-4) and observed high selectivity for CRBN in all cell lines. Furthermore, we directly compared our compounds to current lead CRBN degraders and demonstrated how these probes can be used as chemical knockdown reagents for studying CRBN-dependent processes. Overall, our work provides a roadmap for thorough degrader characterization by combination western and proteomic analysis, as illustrated by the identification of ZXH-4-130 and ZXH-4-137 as CRBN-knockdown tool compounds suitable for cell-based studies.

  View details for DOI 10.1039/d0md00382d

  View details for PubMedID 34458741

  View details for PubMedCentralID PMC8372211

• Exploring Ligand-Directed N-Acyl-N-alkylsulfonamide-Based Acylation Chemistry for Potential Targeted Degrader Development. ACS medicinal chemistry letters Teng, M., Jiang, J., Ficarro, S. B., Seo, H., Bae, J. H., Donovan, K. A., Fischer, E. S., Zhang, T., Dhe-Paganon, S., Marto, J. A., Gray, N. S. 2021; 12 (8): 1302-1307

  Abstract

  Ligand-directed bioconjugation strategies have been used for selective protein labeling in live cells or tissue samples in applications such as live-cell imaging. Here we hypothesized that a similar strategy could be used for targeted protein degradation. To test this possibility, we developed a series of CDK2-targeting N-acyl-N-alkylsulfonamide (NASA)-containing acylation probes. The probes featured three components: a CDK2 homing ligand, a CRL4CRBN E3 ligase recruiting ligand, and a NASA functionality. We determined that upon target binding, NASA-mediated reaction resulted in selective functionalization of Lys89 on purified or native CDK2. However, we were unable to observe CDK2 degradation, which is in contrast to the efficient degradation achieved by the use of a structurally similar reversible bivalent degrader. Our analysis suggests that the lack of degradation is due to the failure to form a productive CDK2:CRBN complex. Therefore, although this work demonstrates that NASA chemistry can be used for protein labeling, whether this strategy could enable efficient protein degradation remains an open question.

  View details for DOI 10.1021/acsmedchemlett.1c00285

  View details for PubMedID 34413960

• Correction: Fragment-based covalent ligand discovery. RSC chemical biology Lu, W., Kostic, M., Zhang, T., Che, J., Patricelli, M. P., Jones, L. H., Chouchani, E. T., Gray, N. S. 2021; 2 (2): 670-671

  Abstract

  [This corrects the article DOI: 10.1039/D0CB00222D.].

  View details for DOI 10.1039/d1cb90008k

  View details for PubMedID 34459829

  View details for PubMedCentralID PMC8341680

• Fragment-based covalent ligand discovery. RSC chemical biology Lu, W., Kostic, M., Zhang, T., Che, J., Patricelli, M. P., Jones, L. H., Chouchani, E. T., Gray, N. S. 2021; 2 (2): 354-367

  Abstract

  Targeted covalent inhibitors have regained widespread attention in drug discovery and have emerged as powerful tools for basic biomedical research. Fueled by considerable improvements in mass spectrometry sensitivity and sample processing, chemoproteomic strategies have revealed thousands of proteins that can be covalently modified by reactive small molecules. Fragment-based drug discovery, which has traditionally been used in a target-centric fashion, is now being deployed on a proteome-wide scale thereby expanding its utility to both the discovery of novel covalent ligands and their cognate protein targets. This powerful approach is allowing 'high-throughput' serendipitous discovery of cryptic pockets leading to the identification of pharmacological modulators of proteins previously viewed as "undruggable". The reactive fragment toolkit has been enabled by recent advances in the development of new chemistries that target residues other than cysteine including lysine and tyrosine. Here, we review the emerging area of covalent fragment-based ligand discovery, which integrates the benefits of covalent targeting and fragment-based medicinal chemistry. We discuss how the two strategies synergize to facilitate the efficient discovery of new pharmacological modulators of established and new therapeutic target proteins.

  View details for DOI 10.1039/d0cb00222d

  View details for PubMedID 34458789

  View details for PubMedCentralID PMC8341086

• Synergistic Anti-Tumor Effect of Combining Selective CDK7 and BRD4 Inhibition in Neuroblastoma. Frontiers in oncology Gao, Y., Volegova, M., Nasholm, N., Das, S., Kwiatkowski, N., Abraham, B. J., Zhang, T., Gray, N. S., Gustafson, C., Krajewska, M., George, R. E. 2021; 11: 773186

  Abstract

  Cyclin-dependent kinases (CDKs) that have critical roles in RNA polymerase II (Pol II)-mediated gene transcription are emerging as therapeutic targets in cancer. We have previously shown that THZ1, a covalent inhibitor of CDKs 7/12/13, leads to cytotoxicity in MYCN-amplified neuroblastoma through the downregulation of super-enhancer-associated transcriptional upregulation. Here we determined the effects of YKL-5-124, a novel covalent inhibitor with greater selectivity for CDK7 in neuroblastoma cells.We tested YKL-5-124 in MYCN-amplified and nonamplified neuroblastoma cells individually and in combination with other inhibitors in cell line and animal models. Cell viability, target validation, effects on cell cycle and transcription were analyzed.CDK7 inhibition with YKL-5-124 did not lead to significant cell death, but resulted in aberrant cell cycle progression especially in MYCN-amplified cells. Unlike THZ1, YKL-5-124 had minimal effects on Pol II C-terminal domain phosphorylation, but significantly inhibited that of the CDK1 and CDK2 cell cycle kinases. Combining YKL-5-124 with the BRD4 inhibitor JQ1 resulted in synergistic cytotoxicity. A distinct MYCN-gene expression signature associated with resistance to BRD4 inhibition was suppressed with the combination. The synergy between YKL-5-124 and JQ1 translated into significant tumor regression in cell line and patient-derived xenograft mouse models of neuroblastoma.The combination of CDK7 and BRD4 inhibition provides a therapeutic option for neuroblastoma and suggests that the addition of YKL-5-124 could improve the therapeutic efficacy of JQ1 and delay resistance to BRD4 inhibition.

  View details for DOI 10.3389/fonc.2021.773186

  View details for PubMedID 35198433

  View details for PubMedCentralID PMC8859926

• PRM-LIVE with Trapped Ion Mobility Spectrometry and Its Application in Selectivity Profiling of Kinase Inhibitors. Analytical chemistry Zhu, H., Ficarro, S. B., Alexander, W. M., Fleming, L. E., Adelmant, G., Zhang, T., Willetts, M., Decker, J., Brehmer, S., Krause, M., East, M. P., Gray, N. S., Johnson, G. L., Kruppa, G., Marto, J. A. 2021

  Abstract

  Parallel reaction monitoring (PRM) has emerged as a popular approach for targeted protein quantification. With high ion utilization efficiency and first-in-class acquisition speed, the timsTOF Pro provides a powerful platform for PRM analysis. However, sporadic chromatographic drift in peptide retention time represents a fundamental limitation for the reproducible multiplexing of targets across PRM acquisitions. Here, we present PRM-LIVE, an extensible, Python-based acquisition engine for the timsTOF Pro, which dynamically adjusts detection windows for reproducible target scheduling. In this initial implementation, we used iRT peptides as retention time standards and demonstrated reproducible detection and quantification of 1857 tryptic peptides from the cell lysate in a 60 min PRM-LIVE acquisition. As an application in functional proteomics, we use PRM-LIVE in an activity-based protein profiling platform to assess binding selectivity of small-molecule inhibitors against 220 endogenous human kinases.

  View details for DOI 10.1021/acs.analchem.1c02349

  View details for PubMedID 34606255